The Micacocidin Production-Related RSc1806 Deletion Alters the Quorum Sensing-Dependent Gene Regulation of Ralstonia pseudosolanacearum Strain OE1-1.

The soil-borne phytopathogenic gram-negative bacterium Ralstonia solanacearum species complex (RSSC) produces staphyloferrin B and micacocidin as siderophores that scavenge for trivalent iron (Fe3+) in the environment, depending on the intracellular divalent iron (Fe2+) concentration. The staphyloferrin B-deficient mutant reportedly retains its virulence, but the relationship between micacocidin and virulence remains unconfirmed. To elucidate the effect of micacocidin on RSSC virulence, we generated the micacocidin productivity-deficient mutant (ΔRSc1806) that lacks RSc1806, which encodes a putative polyketide synthase/non-ribosomal peptide synthetase, using the RSSC phylotype I Ralstonia pseudosolanacearum strain OE1-1. When incubated in the condition without Fe2+, ΔRSc1806 showed significantly lower Fe3+-scavenging activity, compared with OE1-1. Until 8 days after inoculation on tomato plants, ΔRSc1806 was not virulent, similar to the mutant (ΔphcA) missing phcA, which encodes the LysR-type transcriptional regulator PhcA that regulates the expression of the genes responsible for quorum sensing (QS)-dependent phenotypes including virulence. The transcriptome analysis revealed that RSc1806 deletion significantly altered the expression of more than 80% of the PhcA-regulated genes in the mutant grown in medium with or without Fe2+. Among the PhcA-regulated genes, the transcript levels of the genes whose expression was affected by the deletion of RSc1806 were strongly and positively correlated between the ΔRSc1806 and the phcA-deletion mutant. Furthermore, the deletion of RSc1806 significantly modified QS-dependent phenotypes, similar to the effects of the deletion of phcA. Collectively, our findings suggest that the deletion of micacocidin production-related RSc1806 alters the regulation of PhcA-regulated genes responsible for QS-dependent phenotypes including virulence as well as Fe3+-scavenging activity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phosphatidylinositol-phospholipase C3 negatively regulates the hypersensitive response via complex signaling with MAP kinase, phytohormones, and reactive oxygen species in Nicotiana benthamiana.

Phospholipid signaling plays important roles in plant immune responses. Here, we focused on two phospholipase C3 (PLC3) orthologs in the Nicotiana benthamiana genome, NbPLC3-1 and NbPLC3-2. We generated NbPLC3-1 and NbPLC3-2-double-silenced plants (NbPLC3s-silenced plants). In NbPLC3s-silenced plants challenged with Ralstonia solanacearum 8107, induction of hypersensitive response (HR)-related cell death and bacterial population reduction was accelerated, and the expression level of Nbhin1, a HR marker gene, was enhanced. Furthermore, the expression levels of genes involved in salicylic acid and jasmonic acid signaling drastically increased, reactive oxygen species production was accelerated, and NbMEK2-induced HR-related cell death was also enhanced. Accelerated HR-related cell death was also observed by bacterial pathogens Pseudomonas cichorii, P. syringae, bacterial AvrA, oomycete INF1, and TMGMV-CP with L1 in NbPLC3s-silenced plants. Although HR-related cell death was accelerated, the bacterial population was not reduced in double NbPLC3s and NbCoi1-suppressed plants nor in NbPLC3s-silenced NahG plants. HR-related cell death acceleration and bacterial population reduction resulting from NbPLC3s-silencing were compromised by the concomitant suppression of either NbPLC3s and NbrbohB (respiratory oxidase homolog B) or NbPLC3s and NbMEK2 (mitogen activated protein kinase kinase 2). Thus, NbPLC3s may negatively regulate both HR-related cell death and disease resistance through MAP kinase- and reactive oxygen species-dependent signaling. Disease resistance was also regulated by NbPLC3s through jasmonic acid- and salicylic acid-dependent pathways.

The behavior of Ralstonia pseudosolanacearum strain OE1-1 and morphological changes of cells in tomato roots.

The soil-borne Gram-negative ß-proteobacterium Ralstonia solanacearum species complex (RSSC) infects tomato roots through the wounds where secondary roots emerge, infecting xylem vessels. Because it is difficult to observe the behavior of RSSC by a fluorescence-based microscopic approach at high magnification, we have little information on its behavior at the root apexes in tomato roots. To analyze the infection route of a strain of phylotype I of RSSC, R. pseudosolanacearum strain OE1-1, which invades tomato roots through the root apexes, we first developed an in vitro pathosystem using 4 day-old-tomato seedlings without secondary roots co-incubated with the strain OE1-1. The microscopic observation of toluidine blue-stained longitudinal semi-thin resin sections of tomato roots allowed to detect attachment of the strain OE1-1 to surfaces of the meristematic and elongation zones in tomato roots. We then observed colonization of OE1-1 in intercellular spaces between epidermis and cortex in the elongation zone, and a detached epidermis in the elongation zone. Furthermore, we observed cortical and endodermal cells without a nucleus and with the cell membrane pulling away from the cell wall. The strain OE1-1 next invaded cell wall-degenerated cortical cells and formed mushroom-shaped biofilms to progress through intercellular spaces of the cortex and endodermis, infecting pericycle cells and xylem vessels. The deletion of egl encoding ß-1,4-endoglucanase, which is one of quorum sensing (QS)-inducible plant cell wall-degrading enzymes (PCDWEs) secreted via the type II secretion system (T2SS) led to a reduced infectivity in cortical cells. Furthermore, the QS-deficient and T2SS-deficient mutants lost their infectivity in cortical cells and the following infection in xylem vessels. Taking together, infection of OE1-1, which attaches to surfaces of the meristematic and elongation zones, in cortical cells of the elongation zone in tomato roots, dependently on QS-inducible PCDWEs secreted via the T2SS, leads to its subsequent infection in xylem vessels.

A study on safe forceps grip force for the intestinal tract using haptic technology.

PURPOSE: The present study used haptic technology to determine the safe forceps grip force for preventing organ damage when handling the intestinal tract. MATERIAL AND METHODS: The small intestines of ten male beagle dogs (weighing 9.5-10 kg) were grasped with the entire forceps for one minute; the small intestines were then pulled out of the forceps and evaluated for damage. The force at which the shaft inside the forceps was pulled to close the tip of the forceps was defined as the grip force. Small intestine damage was classified into macroscopic (serosal defects, hemorrhage, hematomas, grip marks) and microscopic (damage layer to the mucosa, submucosa/muscularis mucosa, inner orbicularis muscle, external longitudinal muscle, serosa/subserosa). Grip marks and damage layer to the serosa/subserosa have been considered as acceptable safety margins when grasping the small intestines of beagle dogs. RESULTS: The macroscopic findings showed that the maximum grip force that produced a 0% incidence of hemorrhage and hematoma was 15 N. At the microscopic level, the maximum grip force that produced a 0% incidence of external longitudinal muscle injury was 15 N, respectively. CONCLUSIONS: A grip force of 15 N does not damage the small intestines of beagle dogs.

The Tn7-Based Genomic Integration Is Dependent on an attTn7 Box in the glms Gene and Is Site-Specific With Monocopy in Ralstonia solanacearum Species Complex.

The Tn7-based genomic integration system enables direct insertion of foreign gene elements into the chromosome downstream of glms in many bacteria species. The glms gene is greatly conserved in Ralstonia solanacearum species complex (RSSC), while its downstream regions are mostly different in the RSSC. Here, we provided genetic evidence to validate that this Tn7 integration is dependent on a conserved 30-bp motif in the glms, called an attTn7 box, and artificial attTn7 boxes elsewhere are competent for the Tn7 integration, which is further confirmed to be simultaneous downstream of both original and artificial attTn7 boxes, using PCR. With the whole-genome resequencing on 500 Tn7-colonies, the Tn7 integration was confirmed to be site- specific at 25 bp downstream of glms with monocopy as a chromosome of the RSSC. Characteristic of a monocopy in a chromosome enables the Tn7-based complementation to fully restore phenotypes of mutants to those of parent strains that are advantageous rather than those based on plasmids with low-copy numbers. The Tn7-based genomic integration system provides a generally applicable and versatile genetic tool for studies of complementation, pathogenesis, overexpression, and in-vivo promoter activity assays with monocopy in the RSSC.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Involvement of a FAD-Linked Oxidase RSc0454 for Expression of the Type III Secretion System and Pathogenicity in Ralstonia solanacearum.

Ralstonia solanacearum RSc0454 is predicted as a FAD-linked oxidase based on protein homologies, while it contains distinct domains of lactate dehydrogenase and succinate dehydrogenase. A previous study demonstrated that RSc0454 exhibits lactate dehydrogenase activity using pyruvate and NADH as substrates, and is essential for pathogenicity of R. solanacearum. Here, we genetically characterized involvement of RSc0454 on bacterial growth and expression of genes for the type III secretion system (T3SS, a pathogenicity determinant) in R. solanacearum. The RSc0454 mutant grew normally in rich medium but grew faintly in host plants, and failed to grow in minimal medium. Supplementary succinate but not lactate could substantially restore some phenotypes of RSc0454 mutants, including faint growth in host plants, diminished growth in the minimal medium, and lost pathogenicity toward host plants. Expression of T3SS genes is directly controlled by a master regulator, HrpB, and hrpB expression is positively regulated by HrpG and PrhG in parallel ways. Deletion of RSc0454 substantially reduced expression levels of hrpB and T3SS both in vitro and in planta. Moreover, RSc0454 is revealed to be required for the T3SS expression via HrpG and PrhG, although through some novel pathway, and impaired expression of these genes was not due to growth deficiency of RSc0454 mutants. RSc0454 is suggested to be important for redox balance inside cells, and supplementary NADH partially restored diminished growth of the RSc0454 mutant in the minimal medium only in the presence of succinate at some moderate concentrations, indicating that the unbalanced redox in the RSc0454 mutant might be responsible for its diminished growth in the minimal medium. Taken together, these results provide novel insights into the understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on expression of the T3SS in R. solanacearum.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Functional Characterization of RsRsgA for Ribosome Biosynthesis and Expression of the Type III Secretion System in Ralstonia solanacearum.

RsgA plays an important role in maturation of 30S subunit in many bacteria that assists in the release of RbfA from the 30S subunit during a late stage of ribosome biosynthesis. Here, we genetically characterized functional roles of RsgA in Ralstonia solanacearum, hereafter designated RsRsgA. Deletion of R. solanacearum rsgA or rbfA resulted in distinct deficiency of 16S ribosomal RNA, significantly slowed growth in broth medium, and diminished growth in nutrient-limited medium, which are similar as phenotypes of rsgA mutants and rbfA mutants of Escherichia coli and other bacteria. Our gene-expression studies revealed that RsRsgA is important for expression of genes encoding the type III secretion system (T3SS) (a pathogenicity determinant of R. solanacearum) both in vitro and in planta. Compared with the wild-type R. solanacearum strain, proliferation of the rsgA and rbfA mutants in tobacco leaves was significantly impaired, while they failed to migrate into tobacco xylem vessels from infiltrated leaves, and hence, these two mutants failed to cause any bacterial wilt disease in tobacco plants. It was further revealed that rsgA expression was highly enhanced under nutrient-limited conditions compared with that in broth medium and RsRsgA affects T3SS expression through the PrhN-PrhG-HrpB pathway. Moreover, expression of a subset of type III effectors was substantially impaired in the rsgA mutant, some of which are responsible for R. solanacearum GMI1000 elicitation of a hypersensitive response (HR) in tobacco leaves, while RsRsgA is not required for HR elicitation of GMI1000 in tobacco leaves. All these results provide novel insights into understanding various biological functions of RsgA proteins and complex regulation on the T3SS in R. solanacearum.

Characterization of Glaciecola sp. enzymes involved in the late steps of degradation of sulfated polysaccharide ulvan extracted from Ulva ohnoi.

Ulvan is a complex water-soluble sulfated polysaccharide in the cell wall of green algae belonging to genus Ulva. It is composed of l-rhamnose-3-sulfate (Rha3S), glucuronic acid (GluA), iduronic acid (IduA), and d-xylose (Xyl) distributed in three repetition moieties. The first step of a bacterial ulvan degradation is the cleavage of the ß-glycosidic bond between Rha3S and GluA/IduA through a ß-elimination mechanism by a ulvan lyase to produce oligo-ulvans with unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronate (Δ) at the non-reducing end. We have identified an ulvan associated polysaccharide utilization locus (PUL) residing between two ulvan lyase genes belonging to families of polysaccharide lyase 24 (PL24) and PL25 in the genome of a ulvan-utilizing bacterium Glaciecola KUL10 strain. The PUL contains many genes responsible for oligo-ulvan degradation. Among them, we demonstrated that both KUL10_26540 and KUL10_26770 had an unsaturated ß-glucuronyl hydrolase activity to produce Rha3S and oligosaccharides, such as Rha3S-GluA-Rha3S, Rha3S-IduA-Rha3S and, Rha3S-Xyl-Rha3S, by releasing 5-dehydro-4-deoxy-d-glucuronate. KUL10_26540 showed much higher activity than KUL10_26770 and was more active on disaccharide than tetrasaccharide. We also found a rhamnosidase activity on four KUL10 gene products, although they could not react on the sulfated rhamnose.

Phosphatidylinositol-phospholipase C2 regulates pattern-triggered immunity in Nicotiana benthamiana.

Phospholipid signaling plays an important role in plant immune responses against phytopathogenic bacteria in Nicotiana benthamiana. Here, we isolated two phospholipase C2 (PLC2) orthologs in the N. benthamiana genome, designated as PLC2-1 and 2-2. Both NbPLC2-1 and NbPLC2-2 were expressed in most tissues and were induced by infiltration with bacteria and flg22. NbPLC2-1 and NbPLC2-2 (NbPLC2s) double-silenced plants showed a moderately reduced growth phenotype. The induction of the hypersensitive response was not affected, but bacterial growth and the appearance of bacterial wilt were accelerated in NbPLC2s-silenced plants when they were challenged with a virulent strain of Ralstonia solanacearum that was compatible with N. benthamiana. NbPLC2s-silenced plants showed reduced expression levels of NbPR-4, a marker gene for jasmonic acid signaling, and decreased jasmonic acid and jasmonoyl-L-isoleucine contents after inoculation with R. solanacearum. The induction of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes was reduced in NbPLC2s-silenced plants after infiltration with R. solanacearum or Pseudomonas fluorescens. Accordingly, the resistance induced by flg22 was compromised in NbPLC2s-silenced plants. In addition, the expression of flg22-induced PTI marker genes, the oxidative burst, stomatal closure, and callose deposition were all reduced in the silenced plants. Thus, NbPLC2s might have important roles in pre- and post-invasive defenses, namely in the induction of PTI.

Specific Reconstruction on pRC Plasmid to Facilitate Its Universal Chromosomal Integration in Different Ralstonia solanacearum Species Complex Strains.

The pRC system is an efficient tool for genetic studies in Ralstonia solanacearum, ensuring direct insertion of foreign gene elements into Ralstonia chromosome downstream of glms. This system is designed for double recombination across glms and the downstream region, which considerably simplifies genetic studies of complementation, overexpression, pathogenicity, and in-vivo promoter activity assays with monocopy in R. solanacearum, one of the most destructive plant-pathogenic bacteria worldwide. R. solanacearum is extremely heterogeneous and is currently referred to as a Ralstonia solanacearum species complex (RSSC). The glms gene is greatly conserved, but its downstream regions are mostly different in the RSSC, which limits the application of the current pRC plasmid in the RSSC. We compared all existing 132 genome sequences in a precise genomic glms downstream region and confirmed that the pRC system is appropriate for application of chromosomal integration in all RSSC strains but needs respective reconstruction on current pRC plasmids, since glms downstream regions are greatly variable in the RSSC. RSSC strains can be grouped according to identical glms downstream regions. This grouping provides valuable information for gene insertion in this chromosomal region, as it facilitates universal application of the pRC system in RSSC strains.

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins.

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

Efficient renaturation of inclusion body proteins denatured by SDS.

Inclusion bodies are often formed when the foreign protein is over expressed in Escherichia coli. Since proteins in inclusion bodies are inactive, denaturing and refolding of inclusion body proteins are necessary to obtain the active form. Instead of the conventional denaturants, urea and guanidine hydrochloride, a strong anionic detergent SDS was used to solubilize C-terminal His-tag form of ulvan lyase in the inclusion bodies. Solution containing SDS-solubilized enzyme were kept on ice to precipitate SDS, followed by SDS-KCl insoluble crystal formation to remove SDS completely. After removing the precipitate by centrifugation, the supernatant was applied to Ni-NTA column to purify His-tagged ulvan lyase. The purified protein showed a dimeric form and ulvan lyase activity, demonstrating that SDS-denatured protein was renatured and recovered enzyme activity. This simple method could be useful for refolding other inclusion body proteins.

Structural Insights into l-Tryptophan Dehydrogenase from a Photoautotrophic Cyanobacterium, Nostoc punctiforme.

l-Tryptophan dehydrogenase from Nostoc punctiforme NIES-2108 (NpTrpDH), despite exhibiting high amino acid sequence identity (>30%)/homology (>50%) with NAD(P)+-dependent l-Glu/l-Leu/l-Phe/l-Val dehydrogenases, exclusively catalyzes reversible oxidative deamination of l-Trp to 3-indolepyruvate in the presence of NAD+ Here, we determined the crystal structure of the apo form of NpTrpDH. The structure of the NpTrpDH monomer, which exhibited high similarity to that of l-Glu/l-Leu/l-Phe dehydrogenases, consisted of a substrate-binding domain (domain I, residues 3 to 133 and 328 to 343) and an NAD+/NADH-binding domain (domain II, residues 142 to 327) separated by a deep cleft. The apo-NpTrpDH existed in an open conformation, where domains I and II were apart from each other. The subunits dimerized themselves mainly through interactions between amino acid residues around the ß-1 strand of each subunit, as was observed in the case of l-Phe dehydrogenase. The binding site for the substrate l-Trp was predicted by a molecular docking simulation and validated by site-directed mutagenesis. Several hydrophobic residues, which were located in the active site of NpTrpDH and possibly interacted with the side chain of the substrate l-Trp, were arranged similarly to that found in l-Leu/l-Phe dehydrogenases but fairly different from that of an l-Glu dehydrogenase. Our crystal structure revealed that Met-40, Ala-69, Ile-74, Ile-110, Leu-288, Ile-289, and Tyr-292 formed a hydrophobic cluster around the active site. The results of the site-directed mutagenesis experiments suggested that the hydrophobic cluster plays critical roles in protein folding, l-Trp recognition, and catalysis. Our results provide critical information for further characterization and engineering of this enzyme. IMPORTANCE: In this study, we determined the three-dimensional structure of l-Trp dehydrogenase, analyzed its various site-directed substitution mutants at residues located in the active site, and obtained the following informative results. Several residues in the active site form a hydrophobic cluster, which may be a part of the hydrophobic core essential for protein folding. To our knowledge, there is no previous report demonstrating that a hydrophobic cluster in the active site of any l-amino acid dehydrogenase may have a critical impact on protein folding. Furthermore, our results suggest that this hydrophobic cluster could strictly accommodate l-Trp. These studies show the structural characteristics of l-Trp dehydrogenase and hence would facilitate novel applications of l-Trp dehydrogenase.

Characterization of an Alteromonas long-type ulvan lyase involved in the degradation of ulvan extracted from Ulva ohnoi.

Ulvan is a sulfated polysaccharide found in the cell wall of the green algae Ulva. We first isolated several ulvan-utilizing Alteromonas sp. from the feces of small marine animals. The strain with the highest ulvan-degrading activity, KUL17, was analyzed further. We identified a 55-kDa ulvan-degrading protein secreted by this strain and cloned the gene encoding for it. The deduced amino acid sequence indicated that the enzyme belongs to polysaccharide lyase family 24 and thus the protein was named ulvan lyase. The predicted molecular mass of this enzyme is 110 kDa, which is different from that of the identified protein. By deletion analysis, the catalytic domain was proven to be located on the N-terminal half of the protein. KUL17 contains two ulvan lyases, one long and one short, but the secreted and cleaved long ulvan lyase was demonstrated to be the major enzyme for ulvan degradation.

Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum.

Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.

Crystal structure of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase, an NAD⁺-dependent dismutase from Mesorhizobium loti.

5-Formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid 5-dehydrogenase (FHMPCDH) from Mesorhizobium loti is the fifth enzyme in degradation pathway I for pyridoxine. The enzyme catalyzes a dismutation reaction: the oxidation of 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid (FHMPC) to 3-hydroxy-2-methylpyridine 4,5-dicarboxylic acid with NAD(+) and reduction of FHMPC to 4-pyridoxic acid with NADH. FHMPCDH belongs to the l-3-hydroxyacyl-CoA dehydrogenase (HAD) family. The crystal structure was determined by molecular replacement and refined to a resolution of 1.55Å (R-factor of 16.4%, Rfree=19.4%). There were two monomers in the asymmetric unit. The overall structure of the monomer consisted of N- and C-terminal domains connected by a short linker loop. The monomer was similar to members of the HAD family (RMSD=1.9Å). The active site was located between the domains and highly conserved to that of human heart l-3-hydroxyacyl-CoA dehydrogenase (HhHAD). His-Glu catalytic dyad, a serine and two asparagine residues of HhHAD were conserved. Ser116, His137 and Glu149 in FHMPCDH are connected by a hydrogen bonding network forming a catalytic triad. The functions of the active site residues in the reaction mechanism are discussed.

Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

Ralstonia solanacearum, a plant pathogenic bacterium causing "bacterial wilt" on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3-hydroxypalmitate (3-OH PAME) was previously identified as the QS signal in strain AW1. However, 3-OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)-3-OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types--(R)-3-OH MAME or (R)-3-OH PAME. These results demonstrate that (R)-3-OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.

Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti.

Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose-methanol-choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His6-tag and PNOX-pyridoxamine (PM) complex were determined at 2.2Å and at 2.1Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1Å from the N4' atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4'-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4' atom in PM is located at 3.2Å, and the hydride ion from the C4' atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His-Asn or His-His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.

Involvement of ralfuranone production in the virulence of Ralstonia solanacearum OE1-1.

Ralstonia solanacearum causes a destructive disease called "bacterial wilt" in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell-density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J-L, new PhcA-regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1-1. Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1-1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1-1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1-1.

Complete genome sequence of Japanese RSSC phylotype-I strains infecting different host plants.

Ralstonia solanacearum species complex (RSSC) shows a broad host range and is classified into four phylotypes. To compare type III effectors, we have determined the complete genome sequences of several RSSC strains, especially phylotype-I strains isolated in Japan, with different host specificity.